Download Uncommon pathways of metabolism among lactic acid bacteria

FEMS Microbiology Reviews 87
(1990)103-112
103
Publishedby Els~ier
FEMSRE 06162
Uncommon pathways of metabolism among lactic acid bacteria
Jack L o n d o n
Laboratory of MicrobialEcology, National Institute of Dental Research, NationalInstitutes of Health, Bethesda, MD, U.S.A.
Key words: Metabolism; Energy Production; Gluconate; Malate; Xylitol; Ribitol
1. SUMMARY
A small number of lactic acid bacteria possess
the ability to derive energy from organic molecules not utilized by the vast majority of representatives of this large group of microorganisms.
Thus, strains of Lactobacillus casei and enterococci readily grow at the expense of substrates such
as gluconate, malate and pentitols. Transport of
gluconate and pentitols is catalysed by phosphotransferase sytems unique to these bacteria.
Similarly, ",he initial steps in peL,titol dissimilation
are mediated by enzymes found only i~ Lb. casei
and Streptococcus avium.
2. INTRODUCTION
Individually, most of the species that comprise
the lactic acid bacteria utiDze a relatively restricted number of organic molecules as carbon
and energy sources. However, taken in their entirety, this group of Gram-positive bacteria which
includes the genera, Lactobacillus, Streptococcus,
Pediococcus, Leuconostoc, Lactococcus and Enter-
ococcus, possess a metabolic po:enfial only slightly
Correspondenceto: J. London,Building30, Room314, Labora-
tory of MicrobialEcology, National Instituteof Dental Research, NationalInstitutesof Health, Bethesda, MD, U.S.A.
less impressive than that round among the enteric
bacteria [1,2]. Collectively, these bacteria have the
ability to metabolize a large r~amber of mono- and
oligosaccharides, polyalcohols, aliphatic compounds, mono-, di- and tricarboxylic acids and
some amino acids. Synthetically, they possess the
potential to manufacture a host of complex
carbohydrates (expressed as cell wall antigens or
loosely associated slime matrices) and, in at least
one instance, some rare amino acids. Past contributions to this series of symposia have dealt with
the general issues of energy metabolism, transport
and metabolic regulation of the more familiar
sugar substrates. This paper will emphasize research on some uncommon and ~musual metabolic
pathways that have been observed in a restricted
number of lactic acid bacteria.
3. GLUCONATE
It is a generally known and widely accepted
observation that many, if not all, strains of Enterococcus (Streptococcus) faecalis readily grow at the
expense of gluconate, althc.agh Bergey's Manual
of Systematic Bacteriology [1] makes no mention
of this property. Many strains of Lactobacillus
casei possess the same trait (J. London, unpublished observations). Studies with E. faecalis [3]
established that growth on gluconate induced a
NAD+-dependent gluconate-6-P dehydrogenase
0168-6445/90/$03.50© 1990Federationof EuropeanMicrobiologicalSocieties
104
(6GPdh) 1 which coexisted with a constitutively
synthesized NADP+-dependent 6GPdh 2 [4].
6GP + NAD + --* RuSP + CO 2 + NADH
(1)
6GP + NADP +-* Ru5P + CO 2 + NADPH
(2)
The latter enzyme was believed to function in a
biosynthetic capacity providing the cell with
NADPH [5] and was subject to negative allosteric
regulation by an intermediate of glucose catabolism, fructose-l,6-diphosphate (FDP). In contrast,
the NAD+-linked 6GPdh appeared to function
exclusively to provide the cells with energy by
substrate phosphorylation via a hexose monophosphate shunt (HMS) pathway (Fig. 1) and was not
affected by FDP. Kinetic experiments revealed
that the activity of the NAD+-linked enzyme was
inhibited by ATP and it was postulated that the
rate of gluconate dissimilation was regulated by
the energy requirements of the cell during growth.
Synthesis of the NAD+-specific 6GPdh was com-
RIBITOL
pletely repressed in the presence of 20 mM glucose.
Subsequently, it was shown that an inducible
glucunate-specific, PEP-dependent phosphotransferase system (PTS) mediated the uptake of the
HMS substrate (Fig. 1; [6]). Complementation experiments with cytosolic and membrane-associated components revealed that the system functioned with a soluble rather than enzyme II for
gluconate (II gnd) associated factor III (IIIgnd).
Preliminary characterization studies revealed that
III gnd was a relatively large molecule with an
estimated molecular mass of 50 kDa. However,
like other factors Ill, the subunit molecular weight
appeared to be 12 kDa. Thus far, this transport
system appears to be unique to strains of enterococci, however, a similar pathway may also exist in
those Lb. casei strains that grow at the expense of
gluconate [7]. It is also a unique mode of transport
in another sense. In those lactic acid bacteria
XYLITOL
c==c
RIBtTOL-5-P
XYLITOL-5-P
NADH
GUJCONATE~
~ ~ ~
"~ 1119~'~
NADH
N A D H ~ ETHANOL
.(~.C02 ~'~
~'~
ACETYbP ~
ACETATE
._~
epim
PK
6-P-GLUCONATE
RIBULOSE-5-P
~ XYLULOSE-5-P
I~
+
GLYCERALDEHYDE-3-P
NAD* NAOH
co,
It ( ~ .
~ " ~ ,
/"
P'~.UVATE
'~f" NADH
\ MAI.
~.~
/PERM
MALATE
Fig. I. Pathways of gluconate, ribito], xylltol (and D-arabitol) and malatc. Abbreviations and symbols: epim, epimeras¢; PK,
phosphoketolase;(1),6-P-81uconatedehydrogenase;(2), ribitol-5-Pdehydrogenase;(3),xylitol-5-Pdehydrogenase;(4), medicenzyme.
105
capable of utilizing HMS pathway sugars, i.e.
ribose, xylose and arabinose, the existence of
specific pentose permeases is inferred from the
presence of substrate-specific kinases detected in
cell free extracts of these bacteria [8].
4. PENTITOLS
The utilization of five-carbon polyols, i.e. ribitol, xylitol and v-arabitol, is a relatively rare property occurring only in a handful of procaryotes
and eucaryotes [9,10]. Strains of Streptococcus
avium grow ~t the expense of the three polyalcohols listed ,~bove while certain strains of Lb.
casei utilize either ribitol or xylitol and ~arabitol.
The enzymatic mechanisms by which these substrates are taken up and the initial steps in their
catabolism are unique and have been observed
only with two species of lactic acid bacteria. Substrate-specific phosphotransferase systems import
ribitol or xyhtol and v-arabitol into the cell converting the substrates to their respective pentitol5-phosphates in the process (Fig. 1; [liD. In the
cell, NAD+-specific dehydrogenases oxidize the
pentitol-5-phosphates to their corresponding
pentulose-5-phosphates [12]. At this point, the
pathways merge with routes usually employed by
the lactic acid bacteria to dissimilate pentoses and
gluconate (Fig. 1).
Mechanistically, the pentitol PTS were very
similar to the hexitol PTS used by a large variety
of microorganisms to translocate mannitol [13-15].
However, the ribitol and xylitol transport systems
were not only highly specific for their respective
substrates, they also failed to import mannltol
[11]. Because the xylitol PTS proved to be readily
amenable to biochemical dissection, studies were
concentrated on characterizing the I l l xt: component of this system. The purified III xtt was a
slightly acidic protein that had a molecular mass
of 12 kDa [16]. Monospecific rabbit antisera prepared against this purified soluble PTS component
reacted with lII ~*l from other strains of lactobacilfi and S. avium in immunodiffusion experiments. This observation suggests that the anti8enic structure (and some portion of the amino
acid sequence) of this molecule have been structurally conserved. It has not yet been determined
which of the five histidine residues found in Lb.
casei III xtl catalyse transfer of the phosphate
moiety form HPr to EII xtj [17,18].
The rtl-5-P and xtl-5-P NAD+-specific dehydrogenases were purified from Lb. casei strains CI
16 and CI 83, respectively [12]. Substrate specificity was established with chemically or enzymatieally synthesized ribitol-5-P (rtl-5-P), xylitol-5-P
(xtl-5-P) and D-arabitol-5-P (atl-5-P). Ribitol-5-P
dehydrogenase (Rtl-5-Pdh 3) reduced NAD + only
in the presence of rtl-5-P while xylitol-5-P dehydrogenase (xtl-5-Pdh4) reduced N A D + with both
atl-5-P and xtl-5-P as substrata.
rtl-5-P + N A D + --, ru-5-P + N A D H
(3)
xtl-5-P + N A D +--* xu-5-P + N A D H
(4)
In the native state, rtl-5-Pdh and xtl-5-Pdh exist as
a dimer and tetramer, respectively, and migrate
like proteins with estimated molecular masses of
115 kDa and 180 kDa. Like the III xt] of this group
of bacteria, immunological cross reactivity between the streptococcal and lactobacillus xtl-5oPdh
was readily demonstrated. However, none was observed between the rtl-5-P and xtl-5-P dehydrogenases in either intra- and intergeneric cross
matches.
Those strains of Lb. casei that grow at the
expense of ribitol simultaneously induce a functional xylitol PTS, however, they have apparently
lost the ability to synthesize a xtl-5-Pa~ and cannot further metabolize the phosphorylated xylitol
intermediate [11,19]. The inability to metabolize
intracellular xtl-5-P had serious consequences for
growing cells. The addition of xyfitol to an actively growing culture (with ribitiol as energy
source) quickly leads to the PEP-dependent accumulation of high levels of non-metabolizable xtl-5P. However, the microorganisms also possess a
phosphohydrolase activity of broad specificity that
acts on a number of phosphorylated intermediate
products including xtl-5-P [20]. The net effect of
these two activities, xyfitol transport and phosphohydrolase, is the creation of a futile cycle that
dissipates the cells' energy by continuously depleting its PEP pool [21]. This effect is manifested by
106
a cessation of growth at the expense of ribitol;
metabolism of this substrate merely primes the
PEP dissipating pump. Ultimately, the cells overcame the xylitol-induced bacteriostasis by repressing synthesis of the EII xtl of the xylitol I r i s
via a mechanism which is not yet understood.
Repression of the Eli xu remained effective only so
long as xylitol was present in the growth medium.
Growth of repressed cells at the expense of ribitol,
but in the absence of xylitol, returned the microorganism to its xylitol-sensitive ground state.
Xylitol-driven futile cycles reported in other
streptococci have been reviewed elsewhere [10].
The action of the xylitol futile cycle is similar to
the futile cycle driven by 2-deoxyglucose in strains
of Streptococcus pyogenes and Lactococcus lactis
[22,23].
Studies with chemically mutated isolates of Lb.
casei strain C183 were used to ascertain how this
microorganism regulates synthesis of the proteins
that comprise the xylitol pathway, namely EII xtl,
lIl xtl and xylitol-5-P dehydrogenate [10]. Preliminary experiments revealed that ribose and gluconate, or their metabolic intermediate products,
served as gratuitous inducers of the xylitol pathway. Two classes of mutations were observed; one
group, which expressed the phenotype x t l - / a t l - ,
was unable to grow at the expense of either xylitol
or D-arabitol. '12ds group consisted of mutants in
which one of the three xylitol pathway components, EII xtl, III xtl or xti-5-Pdh, was not synthesized in an active form and these isolates were
classified as structural gene mutants. The second
major class of mutants exhibited one of the following phenotypes: x t l - / a t l +, xtl+/atl - or
x t l - / a t l - . The isolates possessed all three components of the pathway when cultivated on ribose or
gluconate and would grow on either pentitol if
sufficient gratuitous inducer was added to the
medium. The multiplicity of inducing substrates,
i.e. xylitol, D-arabitol, ribose and gluconate, and
the L~olation of the three phenotypic expression
groups described above was rationalized by postulating that the cells' genome contained three
regulatory genes whose products function in a
positive fashion upon interacting with xtl-5-P, atl5-P or xu-5-P.
5. MALATE
With few exceptions, most lactic acid bacteria
are capable of catalysing a malolactate fermentation5 in which malic acid (a dicarboxylic acid) is
converted to lactate [24,25].
malate - , lactate + CO 2
(5)
Although the reaction is generally thought to be
non-energy yielding process, the export of lactate
in symport with proton(s) may establish an energy
yielding proton motive gradient across the cells'
membrane similar to that described with Lacwcoccus lactis ssp. cremoris [26,27]. Several malolactic enzymes have been either partially purified
or purified to homogeneity and characterized.
Studies with the partially purified preparation
from Leuconostoc mesenteroides [28] and the pure
preparation from Lb. plantarum [25] indicated
that the malolactate fermentation is not catalysed
by a heterogeneous enzyme complex performing a
variety of individual steps, but rather that a single
NAD+-dependent protein mediates the entire reaction. Because free NADH is not produced by
the Lb. plantarum enzyme, the reaction appears to
be fundamentally decarboxylative in nature
[25,28]. Immunological studies with anti-Lb.
plantarum malie enzyme suggests that the antigenic structure of the protein is conserved within
lactic acid bacteria. Recently, a L, iactis gene
encoding a protein that acts as a positive regulator
for inductic,i~ of the malolaedc enzyme was described [29]. Introduction of the cloned gene into
mutants that were incapable of catalysing the
malolactic reaction but that still possessed both
the malate permease system and malolactic enzyme, restored activity completely.
Strains of E. faecalis and Lb. casei differ from
other lactic acid bacteria by virtue of their ability
to utilize L-malate as a growth substrate. Studies
established that this dicarboxylic TCA cycle intermediate product was dissimilated by an inducible
system consisting of a specific permease which
transported malate into the cell and a NAD +specific 'malic enzyme' (eqn. 6) that oxidized the
substrate to pyruvate and carbon dioxide [30,31].
107
malate + NAD + - , pyruvate + CO 2 + NADH
(6)
Energy was presumably derived from the conversion of pyruvate to acetate and carbon dioxide.
Aerobically, acetate was the major end product
while anaerobic growth resulted in the production
of almost equimolar amounts of acetate and
ethanol.
Synthesis of malate permease and mafic enzyme were repressed by the addition of glucose at
concentrations greater than 5 mM. However, the
addition of 10 mM glucose to resting cells had no
effect on the transport of the dicarboxylic acid
suggesting that neither components of the glucose
PTS nor intermediate products of the EmbdenMeyerhof pathway affected the activity of this
extant process, i.e. that glucose prevented the operation of the permease [32]. However, electrophorefically homogeneous malic enzyme isolated
from both E. faecalis and Lb. casei was inhibited
by Embden-Meyerhof pathway intermediates FDP
and 3-phosphoglycerate as well as ATP [33,34].
Inhibition by the two intermediate products was
believed to provide a means of regulating carbon
flow, energy production and NADH levels in the
presence of two substrates like glucose and malate.
Inhibition by ATP supposedly furnished the
malate pathway with a means regulating energy
production and carbon flow. Malic enzyme from
both microorganisms exhibited very similar physical and kinetic properties and shared a relatively
high degree of immunological homology [35]. It
would be of interest to learn whether a single
permease imports malate for both the malolactic
and mallc enzymes.
6. LACTATE
The lactic acid bacteria are defined, in part, by
their ability to produce lactate from fermentable
or oxidizable substrates. Yet, some members of
this group are capable of using lactate as a source
of energy; and enterococci exhibit a low, but
measurable, amount of growth at the expense of
this metabolic product [36]. One of these
organisms, a strain of E. faecalis, produces a
lactate oxidase activity when cultivated on a
variety of substrates under oxidative conditions.
Expression of lactate oxidase activity was greatest
when glycerol and pyruvate were used .as growth
substrates. Glucose and gluconate permitted induction of only one-fifth of the enzyme activity
observed with the three carbon compounds while
growth on ribose resulted in the synthesis of a
level of lactate oxidase between that observed with
the three- and six-carbon substrates. For reasons
that are still not clear, fructose completely repressed expression of the lactate oxidase activity.
Although other species of streptococci appear to
be capable of metabolizing lactate [37] or deriving
energy from lactate symport [26,38], growth at the
expense of lactate per se is limited to the one
report cited above.
7. CARBOXYETHYL AMINO ACIDS
Recently, a most interesting series of papers
have appeared in the literature which further attests to the metabolic diversity among the lactic
acid bacteria. Strains of lactococci have been
shown to produce unusual amino acid derivatives
from ornithine and lysine. Analysis of the intraceUular amino acid pool of Lactococc~.s lactls
strain 133 during growth in 'spent' medium, revealed high levels of a neutral compound, tentatively identified as ' valine' [39]. This new amino
acid, which was produced concomitant with the
conversion of arginine to ornithine by cultures of
L lactis, was subsequently isolated and identified
as NS-(carboxyethyl)-ornithine [40]. Studies with
L lactis strain K1 that were designed to elucidate
the mechanism by which NS-(carboxyethyl)ornithine was synthesized led to the isolation and
identification of a second hitherto unknown amino
acid, N6-(carboxyethyl)-lysine [41]. Fig. 2 shows
the formulation for the synthesis and structure of
N5-(carboxylethyl) ornithine (the lysine derivative
has a similar structure). Naturally occurring
carboxyethyl derivatives of amino acids are rare in
nature (for review see [42]). Within the biosphere,
108
they appear, thus far, to be produced only by
certain marine invertebrates [~3,44] and constitute
the opines produced in crowri gall of plants infected by species of Agrobacterium [40,42].
Biosynthesis of the carboxyethyl derivatives of
ornithlne and lysine occurs by a relatively simple
NADPH-dependent reductive condensation between the respective amino acid and pyruvate [45].
Both reactions are catalysed by a single enzyme,
a NADP+-specific NS-(L-l-carboxyethyl)-L-orni
thine oxidoreductase (EC. 1.5.1.24). The native
enzyme is a tetramer consisting of four 38-kDa
subunlts and is present in the cell at very low
concentrations [45]. At present, the 'synthase' appears to be restricted to strains of L. lactis and
has not been observed in extracts of other species
of lactic acid bacteria [46]. Although the enzyme
can provide a means of reoxidizing NADPH, this
may not be its primary function in the cell. An
opine dehydrogenase of relatively broad specificity produced by a species of Arthrobacter condenses the amino acids methionlne and isoleucine
and with pyruvate to form the corresponding N[1-R-(carboxy)ethyl] derivatives [47]. However,
since this strain of Arthrobacter grows at the expense of opines, presumably of plant origin, the
function of the enzyme appears to be degradative
rather than biosynthetic. The function of the
lactococcal enzyme and the role(s) of the carboxethyl derivatives of ornithine and lysine in this
organism's metabolism have yet to be established.
8. REGULATION OF MALATE AND PENTITOL METABOLISM BY LACTIC ACID BACTERIA: CONUNDRUMS A N D RATIONALIZATIONS
The fine control systems observed in Staphylococcus aureus [48] and Gram-negative bacteria [49]
which inhibit transport and metabolism of glycerol
or glycerol and lactate metabolism, respectively,
when a Errs sugar is present do not appear to
operate so effectively in those lactic acid bacteria
that metabolize pentitols and malate. Growing
cultures of Lb. casei and E. faecalis have been
observed to utilize pentitols and glucose simultaneously, while resting cells of E. faecalis transport
malate in the presence of bexose. The transport of
pentitols and malate is mediated by a I r i s and
permease, respectively, yet their uptake by growing or resting cell suspensions was virtually unaffected by the addition of 10 mM glucose [19,20].
Uptake and, in the case of the pentitols, ntilization of these substrates, ceased only after several
generations of growth in the presence of glucose.
These data suggest that some glycolytic intermediate triggers the formation of a repressor of
biosynthesis of both the transport enzymes and
dehydrogenases. In time, the various activities were
diluted to the point where they became non-functional. A similar phenomenon was observed during the operation of the xylitol futile cycle in Lb.
casei cells in which the pentitol transport system
o
~
-
o~
Omithine
H H
H÷
H~O
~
_
~
?ls.(L.l.carboxyethyl)L-omithine
Fig. 2. Structureof NS-(carboxyethyl)omithineand equationdescribingthe synthesisof the aminoacid derivative(figurecourtesyof
Dr. J. Thompson).
109
drained away significant amounts of the cells'
energy supply generated during growth on glucose
[20]. Glucose (10 mM) had no apparent effect on
xylitol uptake and the functioning futile cycle
(which consists of the xylitol PTS and a phoso
phohydrolase) reduced the growth rate markedly.
Again, it appears that the primary source of control of the futile cycle enzymes is exerted at the
level of transcription because the inhibitory effects
of xylitol were not overcome during glucose-supported growth over the course of the experiment
[20] and growth at the expense of ribitol was
inhibited for as long as several days [19].
The xylitol-5-P dehydrogenase also remained
detectable and functional in cells long after glucose had been added to the culture. However,
malic enzyme was sensitive to glycolytic intermediates and conversion of malate to pyruvate
and CO2 could theoretically be inhibited by intermediate products of glucose catabolism [32]. The
ribitol-5-P, xylitol-5-P and 6-phosphogluconate
dehydrogenases differ from malic enzyme in that
they are not subject to allosteric inhibition by
glycolytic intermediates. The NAD +-linked malic
enzyme clearly belongs to that of the group of
catabolic and biosynthetic enzymes under the control of specific glycolytic intermediates. It would
appear that carbon channeling from gluconate,
xylitol or ribitol into the Embden-Meyerhof pathway at the level of triose phosphate and acetyl
phosphate is readily accommodated by streptococcal or lactobacillus cells metabolizing hexose
simultaneously. This is probably due to the fact
that only half of the carbon from ribitol and 25~
of the carbon from xylitol is processed via LDH.
Moreover, excess reducing equivalents can be directed into the production of ethanol under
anaerobic conditions. This does not appear to be
the case for carbon derived from malate which
enters the glycolytic pathway at the level of pyruvate. In the absence of an external electron acceptor like oxygen, the malate-derived pyruvate may
compete with the pyruvate originating from glucose for a limited number of reducing equivalents
and upset the ceils' metabolic balance. However,
certain lactic acid bacteria apparently can coordinate the efficient utilization of two substrates
simultaneously, at least for limited periods of time.
This property may endow these bacteria with a
selective advantage over more rigorously regulated
bacteria in an environment rich in organic nutrients.
9. COI~ICLUDING REMARKS
One of the primary purposes of this presentation was to review some of the rarer metabolic
traits and pathways of lactic acid bacteria that had
been defined biochemically and enzymatically over
the past 20 years. The genetic system responsible
for the biosynthesis of the carbox-yethyl amino
acid derivatives is currently under vigorous investigation at the National Institute of Dental Research. This is not the case for the gluconate,
pentitol or malate dissimilatory systems. Probes
are either available or can readily be obtained for
cloning the genes which encode proteins of the
latter two systems. Such studies are vital to understanding how these genes are arranged and how
transcription is regulated in the lactic acid bacteria.
It is hoped that this treatise will serve as a reminder and impetus for future investigations of
these pathways 'less travelled' in the lactic acid
bacteria.
ACKNOWLEDGEMENTS
This paper is dedicated to my friend and colleague, Morrison Rogosa, who died March 28,
1989. His fife-long devotion to defming the lactic
acid bacteria inspired his co-workers. I am also
grateful to Drs. John Thompson, Paul Kolenbrander and Stan Robrish for their helpful criticism and comments.
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Purification and properties of a malolactic enzyme from
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are novel imino acids produced by a dchydrogenase found
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in Streptococcus lactis: distribution, constitutivity, and
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Regulation of carbohydrate uptake and adenylate cycAase
activity mediated by th~ enzymes I1 of the phosphoenolpyruvate:sugar phosphate system in Escherichia coil J.
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